| Objective: Hydrogen sulfide(H2S) is a kind of pungent toxic gases and environmental pollution. However, recent studies have shown that H2 S is the third signaling molecules second of NO and CO, which can freely pass through the cell membrane, play the physiological and pathological role in mammalian body tissues and cells as neuromodulator, so get wide attention by scientific community. This study aimed to establish a quantitative analysis method for determining H2 S in rat intestinal perfusate which was sensitive, rapid and online and provide a reference basis and implementation case for effective determination of H2 S in mammals.Methods: Using Na2 S as donor of H2 S, combined optical fiber sensor technology with Micro Sequential Injection lab-on-valve(μSIA- LOV), under conditions of that the sample volume was 50 μL, using calcein-copper(Ⅱ) fluorescence recovery method, mercury fluorescein fluorescence quenching method and methylene blue method to determine H2 S in rat intestinal perfusate. To explore and optimizing concentration of reagent, dosage of reagent, p H for reaction, time of reaction, stability of product and detection flow rate.Results:(1) Calcein-copper(Ⅱ) system fluorescence recovery method: 200μL phosphate buffer as the carrier(p H=8.0), 1.0×10-5mol/L calcein 50 μL, 1.0×10-5 mol/L copper sulfate 50 μL, the linear range to determine H2 S in rat intestinal perfusate was 1.0×10-5mol/L ~ 6.0×10-5mol/L, sample volume was 50 μL, detection flow rate was 30μL/s, RSD=3.3%, the detection limit was 5.4×10-6mol/L, concentration of H2 S in sample was 4.1×10-5mol/L, the average recovery was 91.1%, the analysis frequency was 40 samples/h.(2) Mercury fluorescein fluorescence quenching method: 100 μL 0.1mol/L Na OH solution as the carrier, 1.0×10-5mol/L fluorescein mercury 50 μL, sample volume was 50 μL, detection flow rate was 25μL/s, the linear range to determine H2 S in rat intestinal perfusate was 5.0×10-6~1.0×10-4 mol/L, RSD=3.8%, the detection limit was 4.1×10-7mol/L, concentration of H2 S in sample was 6.6×10-5mol/L, the average recovery was 98.2%, the analysis frequency was 40 samples/h.(3) methylene blue method: under acidic conditions, with 100μL water as carrier, 0.05mol/L zinc acetate 50μL, 1.0×10-3mol/L N,N- dimethyl-p- phenylenediamine hydrochloride 50μL, 0.1 mol / L of Fe Cl3 50μL, absorbance of methylene blue was measured at 663.49 nm, sample volume was 50 μL, detection flow rate was 5μL/s, the linear range to determine H2 S in rat intestinal perfusate was 2.0×10-5~4.0×10-4mol/L, RSD=3.4%, the detection limit was 4.7×10-6mol/L, concentration of H2 S in sample was 1.62×10-4mol/L, the average recovery was 101.7%, the analysis frequency was 20 samples/h.Conclusion: Combine optical fiber sensor technology with μSIA- LOV system, developing both of their features and advantages,to determine H2S’s content in rat intestinal perfusate quickly and accurately by using methylene blue method and fluorescence analysis technology, reach the intended purpose. Not only to provide a technical reference of application for optical fiber sensing technology and μSIALOV, but also laid the foundation of determining H2 S in biological samples sensitively, rapidly and online. |
